Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
Several methods are available to monitor gene activity and protein distribution within cells. These include the formation of fusion proteins with coding sequences for .beta.-galactosidase (21), and luciferases (21). The usefulness of these methods is often limited by the requirement to fix cell preparations or to add exogenous substrates or cofactors. This invention disclose a method of examining gene expression and protein localization in living cells that requires no exogenously-added compounds.
This method uses a cDNA encoding the Green-Fluorescent Protein (GFP) from the jelly fish Aequorea victoria (5). In A. victoria, GFP absorbs light generated by aequorin upon the addition of calcium and emits a green light.
This invention discloses that GFP expressed in prokaryotic and eukaryotic cells is capable of producing a strong green fluorescence when excited near UV or blue light. Since this fluorescence requires no additional gene products from A. victoria, chromophore formation is not species specific.